Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0393754 (HSA)
 2,996 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three geometric ortho-, meta-, and para-isomers of N-(aminobenzoyloxy)succinimide (ABS) were synthesized, and their usefulness as a two-level heterobifunctional cross-linking agent in the preparation of hapten-protein conjugates was evaluated. The conjugation was based on the principle that ABS reacts immediately with an amino group of a hapten, and an aminobenzoyl group incorporated into the hapten is then activated by diazotization to a functional diazobenzoyl group acting on tyrosine or histidine residues of the protein. Using the anti-tumor antibiotic daunomycin (DM) as a model hapten, the three isomers of ABS were compared for their ability to conjugate DM with bovine serum albumin (BSA); DM incorporation onto a BSA molecular was found to occur to the highest degree with m-ABS, followed by p-ABS. while o-ABS completely failed to conjugate under the same coupling conditions. Using m-ABS it was possible to introduce more than 10 molecules of DM per BSA molecule. One of the DM-BSA samples was used as the immunogen for the production of anti-DM serum in a rabbit. The antibody specificity was shown to be direct to DM but not to other anti-cancer drugs (bleomycin, mitomycin C, actinomycin D and 5-fluorouracil) by the double antibody enzyme immunoassay (DEIA) using DM-beta-galactosidase conjugate as a label. An enzyme-linked immunosorbent assay (ELISA) for anti-DM IgG was developed using a DM-human serum albumin (DM-HSA) conjugate similarly prepared with m-ABS and horseradish peroxidase-conjugated goat anti-rabbit IgG as the solid-phase antigen and the labelled second antibody, respectively. This ELISA permitted us to measure accurately as little as 50 ng of anti-DM IgG per ml using a standard anti-DM IgG which had been purified from the anti-DM serum using an affinity column of Sepharose 4B with DM-HSA as the ligand. Using this ELISA as well as a sandwich enzyme immunoassay (SEIA) for total IgG, serum levels of anti-DM IgG and total IgG levels were easily monitored in a rabbit following immunization with DM-BSA. These results indicate that the use of DBS provides a novel method for preparing hapten-protein conjugates which will be useful in biochemistry and immunochemistry.
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PMID:The use of N-(aminobenzoyloxy) succinimide as a two-level heterobifunctional agent for the preparation of hapten-protein conjugates. Daunomycin as a model hapten with an amino group. 225 68

Recent scanning electron microscopic studies on isolated follicular dendritic cells (FDC) showed that dendrites of certain FDC were "beaded," i.e., consisting of a series of interconnected immune complex coated bodies (termed "iccosomes," measuring 0.3 to 0.7 micron diameter). In vitro these iccosomes detach from one another with ease. The major objectives herein were to establish whether these structures can be detected in sections and whether iccosomes serve to disseminate antigen in vivo. Beginning at day 1, the time point used for isolating beaded FDC, the popliteal lymph nodes of immune C3H mice were studied with light and transmission electron microscopy for 2 wk (i.e., at days 1, 3, 5, 8, and 14) after hind footpad injection of the histochemically detectable antigen, horseradish peroxidase (HRP). Iccosomes (0.25 to 0.38 micron diameter), contoured by a peroxidase (PO)-positive coat of HRP-anti-HRP complexes, were first detected by transmission electron microscopy at day 1 adjacent to cell bodies of certain FDC. Within their limiting membrane they contained flocculent material that was PO positive. At day 3 by light microscopy, germinal centers were seen enlarged and the antigen-retaining reticulum, composed of antigen-bearing FDC, appeared diffuse. This coincided with the transmission electron microscopic visualization of a dispersed state of iccosomes among the follicular lymphocytes. At that time iccosomes were seen attached to the surface of lymphocytes via PO-positive immune complexes and were surrounded by microvillous processes of these cells. Germinal center lymphocytes and tingible body macrophages both responded to contact with iccosomes by endocytosis. Antigen-containing tingible body macrophage were most conspicuous by light microscopy at day 5, when transmission electron microscopy showed that the majority of germinal center lymphocytes contained endocytosed HRP in secondary lysosome-like granules associated with the Golgi apparatus. The number of dispersed iccosomes was markedly reduced by day 5. In controls injected with HSA, a PO-negative antigen, lymphocytes and tingible body macrophages were PO-negative. The presence of antigen in both cell types was confirmed through the use of a gold-conjugated antigen (goat IgG). Simultaneous immunoperoxidase labeling of the same tissues with anti-Ia showed the gold conjugate containing B cells to be Ia+. Antigen-positive B cells and tingible body macrophages were greatly reduced in numbers by day 14, suggesting the intracellular fragmentation of the antigen.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigen-processing cells. 325 33

The enzymic and non-enzymic formation of protein-arylating intermediates from amodiaquine (AQ,7-chloro-4-(3'-diethylamino-4'-hydroxyanilino) quinoline), an anti-malarial associated with agranulocytosis and liver damage in man, was studied in vitro. [14C]AQ in phosphate buffer, pH 7.4, under air was autoxidized to a reactive derivative(s) which possessed characteristics indicative of a semiquinone/quinone imine: reduction by NADPH and ascorbic acid, conjugation with thiols and irreversible binding to microsomal and soluble proteins. Cysteinyl SH groups were major sites of arylation. Radiolabelled material irreversibly bound to HSA after 24 hr and to human liver microsomes after 4 hr represented 26.5 +/- 1.8% and 31.4 +/- 0.6% (means +/- SD, N = 3) of incubated [14C]AQ (10 microM), respectively. The quinone imine of AQ(AQQI) was synthesized, and displayed the same oxidative and electrophilic reactions as the product(s) of AQ's autoxidation. A water-soluble product formed in buffered solutions of AQ and N-acetylcysteine was identified as an AQ mercapturate by comparison with an adduct prepared from synthetic AQQI. Irreversible binding of [14C]AQ was inhibited by a radical scavenger; this indicated that the semiquinone imine contributed to the binding. Although AQ was extensively de-ethylated by human liver microsomes, oxidation by cytochrome P-450 did not appear to be principally responsible for its activation and irreversible binding in microsomal incubations. AQ was oxidized to protein-arylating intermediates by horseradish peroxidase. It also formed reactive derivatives, possibly N-chloro compounds, in chlorine solutions. These findings indicated that AQ can give rise to chemically reactive species by at least three distinct mechanisms, viz. autoxidation in neutral solution under air, peroxidase-catalyzed oxidation and N-chlorination. Formation of such species in liver and myeloid cells might be responsible for the adverse reactions associated with AQ.
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PMID:Drug-protein conjugates--XIV. Mechanisms of formation of protein-arylating intermediates from amodiaquine, a myelotoxin and hepatotoxin in man. 334 86

A sensitive enzyme immunoassay for cortisol in plasma and saliva without extraction has been developed. An antibody was raised in rabbits against cortisol-3-CMO-BSA and compared with anti-cortisol-21-HS-HSA. The enzyme label was a covalently linked cortisol-horseradish peroxidase conjugate. Solvent extraction was avoided by use of heat denaturation of transcortin and dilution of plasma 1:300. This assay had a limit of detection of 3 fmol/tube and satisfied the standard criteria of accuracy and precision. The test requires only a simple photometer. The normal values of cortisol in plasma and saliva before and after ACTH, dexamethasone or metyrapone application were determined. Saliva, being easily collected by a noninvasive, stress-free technique is an alternative to plasma. The cortisol concentrations were significantly increased in plasma and saliva of patients with Cushing's syndrome and decreased in patients with Addison's disease.
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PMID:A sensitive direct enzyme immunoassay for cortisol in plasma and saliva. 609 Jan 83

Cells producing specific antibodies against the hapten penicilloyl (Pen) are demonstrated in tissue sections of the spleen, using peroxidase immunocytochemistry. Horseradish peroxidase-human serum albumin-penicilloyl (HRP-HSA-Pen) conjugate, prepared by coupling Pen to HRP-HSA conjugate, was used for detection of anti-Pen producing cells in the spleen after intravenous injection of bovine gamma globulin-penicilloyl (BGG-Pen) conjugate. HRP-HSA conjugate was used as a control to exclude the possibility that positive cells contained antibody against a common determinant in both HSA and BGG. The method may be applied for the detection of cells producing antibodies against other haptens as well.
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PMID:Peroxidase immunocytochemistry for the detection of specific anti-hapten (penicilloyl) antibody producing cells in the spleen, after injection of a hapten-carrier conjugate. 620 28

A simple, sensitive and direct spectrophotometric method is presented for the determination of epsilon-amino groups of L-lysine present in carrier proteins, using the free amino acids L-lysine and L-glutamic acid as reference standards, and 2,4,6-trinitrobenzene 1-sulfonic acid (TNBS) reagent. The amount of epsilon-amino group present in the carrier protein after coupling with hapten is directly quantitated using the standard curve generated by the difference in absorbance observed with L-lysine and L-glutamic acid after their reaction with TNBS reagent. Spectral analysis of L-lysine and L-glutamic acid (27.36 nmol) derivatives of TNBS at 335 nm showed that TNP-L-lysine had twice the absorbance of TNP-L-glutamic acid, since TNBS reagent interacts equally with the alpha-amino and epsilon-amino groups present in L-lysine and the alpha-amino group of L-glutamic acid, respectively. The relationship between absorbance and concentration of epsilon-amino groups (up to 16 micrograms/ml) was found to be linear. The number of epsilon-amino groups of lysine present in carrier proteins such as BSA, HSA, thyroglobulin and the enzyme, horseradish peroxidase, were analyzed by the present method and were found to be similar to the reported values. Various carrier protein-hapten conjugates (protein-mycotoxin/vitamin/steroid hormone conjugates) were made and analyzed by the method developed in order to determine their mole to mole ratio.
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PMID:Quantitation of epsilon-amino group using amino acids as reference standards by trinitrobenzene sulfonic acid. A simple spectrophotometric method for the estimation of hapten to carrier protein ratio. 790 97

The use of a two-level heterobifunctional agent N-(m-aminobenzoyloxy)succinimide (m-ABS) allowed us to develop a new method for preparing hapten-protein conjugates. This was demonstrated by a conjugation between thyrotropin-releasing hormone (TRH) and bovine or human serum albumin (BSA or HSA). The conjugation is based on the principle that the succinimidyl ester group of m-ABS immediately acts on an epsilon-amino group of lysine residues of carrier protein BSA (or HSA) and a m-aminobenzoyl group incorporated into the protein is then activated by diazotization to a functional m-diazobenzoyl group (m-DB) acting on a histidyl group of TRH. The TRH-BSA containing about 3.5 mol of TRH per BSA molecule, elicited the production of TRH antibody in rabbits. A new type of enzyme-linked immunosorbent assay (ELISA) for TRH was developed using the antiserum, the solid-phase antigen TRH-HSA and the commercially available horseradish peroxidase-labeled goat anti-rabbit IgG/Fab' as a marker, revealing that the ELISA was monospecific to the hormone and measured as low as 50 pg of the hormone reproducibly. Also, using the antiserum by the indirect immunoperoxidase method the distribution of immunoreactive TRH in the rat brain was demonstrated in neurons of the paraventricular nucleus and neuronal processes of the median eminence. These results strongly suggested that the use of m-ABS provided a simple and efficient new method for preparing immunogens not only for the previously reported haptens with a primary amino group(s) (J. Immunol. Methods 134 (1990) 227), but also for haptens with an imidazole, phenolic, or indole group(s) in the molecule.
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PMID:New hapten-protein conjugation method using N-(m-aminobenzoyloxy) succinimide as a two-level heterobifunctional agent: thyrotropin-releasing hormone as a model peptide without free amino or carboxyl groups. 793 Jun 35

Competitive binding assays have been performed in flow injection systems. To further increase the versatility of the system, and to enable it to deal with samples containing particulate matter, the adsorption step was designed as an expanded bed column. Immunochemical quantification of human serum albumin was chosen as a model system to use for the development of the technology. A competitive ELISA was set up using peroxidase labelled HSA as competing ligand. The introduction of the expanded bed immunosorption column made the system tolerant to samples containing suspended particulate matter. The analytical outcome is very similar to that from the packed bed system even though more time is required for each assay cycle. The capability of the system was tested by addition of increasing amounts of yeast cells. The results clearly indicate that the system is suitable e.g. for process monitoring of fermentations.
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PMID:Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column. 1073 76

The size and charge-selective properties of the glomerular barrier are partly controversial. Glomerular sieving coefficients (theta) for proteins have rarely been determined noninvasively before in vivo. Therefore, theta was assessed vs. glomerular filtration rate (GFR; (51)Cr-EDTA clearance) in intact rats for radiolabeled myoglobin, kappa-dimer, neutral horseradish peroxidase (nHRP), neutral human serum albumin (nHSA), and native albumin (HSA). To obtain theta, glomerular tracer clearance, assessed from the 7- to 8-min kidney uptake of protein, was divided by the GFR. The data were fitted with a two-pore model of glomerular permeability, where the small-pore radius was 37.35 +/- 1.11 (SE) A, and the "unrestricted pore area over diffusion path length" (A(0)/DeltaX) 1.84 +/- 0.43 x 10(6) cm. Although seemingly horizontal for nHRP and nHSA, the log theta vs. GFR curves showed slightly negative slopes for the proteins investigated in the GFR interval of 2-4.5 ml/min. Strong negative (linear) correlations between (log) theta and GFR were obtained for myoglobin (P = 0.002) and HSA (P = 0.006), whereas they were relatively weak for nHRP and nHSA and nonsignificant for kappa-dimer. Theta for nHSA was markedly higher than that for HSA. In conclusion, there were no indications of increases in theta vs. GFR, as indicative of concentration polarization, for the proteins investigated at high GFRs. Furthermore, the glomerular small-pore radius assessed from endogenous (neutral) protein sieving data was found to be smaller than previously determined using dextran or Ficoll as test molecules.
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PMID:Glomerular filtration rate dependence of sieving of albumin and some neutral proteins in rat kidneys. 1262 Sep 29

The kinetics of Coprinus cinereus peroxidase-catalyzed 1-naphthol, 2-naphthol, and 4-hydroxybiphenyl oxidation was investigated. The initial rates of the naphthols' and 4-hydroxybiphenyl oxidations were linearly dependent on enzyme concentration. The rates depended on substrate concentration and saturated at concentrations above 100 microM of hydrogen peroxide, 25-50 microM of naphthols, and 10 microM of 4-hydroxybiphenyl. At the peroxide concentration 100 microM calculated K(m) and the maximal rate (V(max)) were 74.7 microM and 0.53 microM/sec or 175 microM and 2.0 microM/sec for 1- or 2-naphthol, respectively, and 29.68 microM and 0.42 microM/sec for 4-hydroxybiphenyl. Kinetic measurements of exhaustive naphthol and 4-hydroxybiphenyl oxidation showed that peroxidase is inactivated during the oxidation of the substrates. Different factors and additives, water soluble polymers and albumins (PEG, PEI, PL, BSA, HSA), influenced the initial naphthols and 4-hydroxybiphenyl oxidation rates, peroxidase inactivation rates, and the degree of the substrate conversion. Addition of albumin increased turnover number of naphthols oxidation 1.5-4 times. Light scattering increase was observed when peroxidase-catalyzed oxidation reaction was investigated and suggested that insoluble particles were formed during the process. The addition of polymers, change of concentration and ionic strength of the solution as well as the number of other factors influenced the observed light scattering. The number of particles formed during peroxidase-catalyzed naphthols' and 4-hydroxybiphenyl oxidation and their distribution according to size in the interval 2.5-300 microm were detected by particle counting in solutions.
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PMID:Oxidation of phenolic compounds by peroxidase in the presence of soluble polymers. 1552 12


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